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Straightforward peptide sequencing by MALDI-MS/MS using a Lys-N metallopeptidase - Boersema et al. 2008

ABSTRACT: Not available

INSTRUMENT(S): instrument model

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Paul Boersema

PROVIDER: PRD000052 | Pride | 2008-12-02

REPOSITORIES: pride

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			Action	DRS
	PRIDE_Exp_Complete_Ac_3380.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_3380.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_3380.xml.gz	Xml

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Publications

Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.

Boersema Paul J PJ Taouatas Nadia N Altelaar A F Maarten AF Gouw Joost W JW Ross Philip L PL Pappin Darryl J DJ Heck Albert J R AJ Mohammed Shabaz S

Molecular & cellular proteomics : MCP 20081129 4

In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applica ...[more]

PMID: 19043101

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Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.

Project description:In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applicability for de novo sequencing we next separated an ostrich muscle tissue protein lysate by one-dimensional SDS-PAGE. A protein band at 42 kDa was in-gel digested with Lys-N. Relatively straightforward sequencing resulted in the de novo identification of the two ostrich proteins creatine kinase and actin. We therefore conclude that this method that combines Lys-N, strong cation exchange enrichment, and MALDI-MS/MS analysis provides a valuable alternative proteomics strategy.

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Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidase

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Reply to Soman et al, Alffenaar et al, Metcalfe et al, and Raoult.

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Peptide Sequencing Directly on Solid Surfaces Using MALDI Mass Spectrometry.

Project description:There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly on surfaces. However, common sequencing approaches that can be adapted to surface bound peptides lack the throughput often needed in library-based applications. Here we describe a simple approach for sequence analysis directly on solid surfaces that is both high speed and high throughput, utilizing equipment available in most protein analysis facilities. In this approach, surface bound peptides, selectively labeled at their N-termini with a positive charge-bearing group, are subjected to controlled degradation in ammonia gas, resulting in a set of fragments differing by a single amino acid that remain spatially confined on the surface they were bound to. These fragments can then be analyzed by MALDI mass spectrometry, and the peptide sequences read directly from the resulting spectra.

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Classification of ancient mammal individuals using dental pulp MALDI-TOF MS peptide profiling.

Project description:BackgroundThe classification of ancient animal corpses at the species level remains a challenging task for forensic scientists and anthropologists. Severe damage and mixed, tiny pieces originating from several skeletons may render morphological classification virtually impossible. Standard approaches are based on sequencing mitochondrial and nuclear targets.Methodology/principal findingsWe present a method that can accurately classify mammalian species using dental pulp and mass spectrometry peptide profiling. Our work was organized into three successive steps. First, after extracting proteins from the dental pulp collected from 37 modern individuals representing 13 mammalian species, trypsin-digested peptides were used for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The resulting peptide profiles accurately classified every individual at the species level in agreement with parallel cytochrome b gene sequencing gold standard. Second, using a 279-modern spectrum database, we blindly classified 33 of 37 teeth collected in 37 modern individuals (89.1%). Third, we classified 10 of 18 teeth (56%) collected in 15 ancient individuals representing five mammal species including human, from five burial sites dating back 8,500 years. Further comparison with an upgraded database comprising ancient specimen profiles yielded 100% classification in ancient teeth. Peptide sequencing yield 4 and 16 different non-keratin proteins including collagen (alpha-1 type I and alpha-2 type I) in human ancient and modern dental pulp, respectively.Conclusions/significanceMass spectrometry peptide profiling of the dental pulp is a new approach that can be added to the arsenal of species classification tools for forensics and anthropology as a complementary method to DNA sequencing. The dental pulp is a new source for collagen and other proteins for the species classification of modern and ancient mammal individuals.

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Identification of potential serum peptide biomarkers of biliary tract cancer using MALDI MS profiling.

Project description:BackgroundThe aim of this discovery study was the identification of peptide serum biomarkers for detecting biliary tract cancer (BTC) using samples from healthy volunteers and benign cases of biliary disease as control groups. This work was based on the hypothesis that cancer-specific exopeptidases exist and that their activities in serum can generate cancer-predictive peptide fragments from circulating proteins during coagulation.MethodsThis case control study used a semi-automated platform incorporating polypeptide extraction linked to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to profile 92 patient serum samples. Predictive models were generated to test a validation serum set from BTC cases and healthy volunteers.ResultsSeveral peptide peaks were found that could significantly differentiate BTC patients from healthy controls and benign biliary disease. A predictive model resulted in a sensitivity of 100% and a specificity of 93.8% in detecting BTC in the validation set, whilst another model gave a sensitivity of 79.5% and a specificity of 83.9% in discriminating BTC from benign biliary disease samples in the training set. Discriminatory peaks were identified by tandem MS as fragments of abundant clotting proteins.ConclusionsSerum MALDI MS peptide signatures can accurately discriminate patients with BTC from healthy volunteers.

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Cryobiopsy Compared with Surgical Lung Biopsy in ILD: Reply to Maldonado et al., Froidure et al., Bendstrup et al., Agarwal et al., Richeldi et al., Rajchgot et al., and Quadrelli et al.

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Reply to Turnbull et al. and to Hulme et al.

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Reply to Topeli et al. and to Akinosoglou et al.

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Reply to Gueret et al. and to Hammond et al.

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